Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
RESEARCH ARTICLE
Open Access
Transforming growth factor beta receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer Bojana Jovanović1, J Scott Beeler2, Michael W Pickup1, Anna Chytil1, Agnieszka E Gorska1, William J Ashby3, Brian D Lehmann2, Andries Zijlstra4, Jennifer A Pietenpol1,2 and Harold L Moses1,4*
Abstract Introduction: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-β) pathway-associated genes relative to other subtypes, including the TGF-β receptor type III (TβRIII). We hypothesize that TβRIII is tumor promoter in mesenchymal-stem like TNBC cells. Methods: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TβRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TβRIII (TβRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TβRIII expression patterns across all TNBC subtypes. Results: TβRIII was the most differentially expressed TGF-β signaling gene in the MSL subtype. Silencing TβRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TβRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TβRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TβRIII-KD. Stable knockdown of integrin-α2 in TβRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. Conclusions: We have found that TβRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TβRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TβRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TβRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.
* Correspondence: [email protected] 1 Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, 2220 Pierce Avenue, 698 Preston Research Building, Nashville, TN 37232, USA 4 Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, 1161 21st Avenue South, C-3321A Medical Center North, Nashville, TN 37232, USA Full list of author information is available at the end of the article © 2014 Jovanović et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
Introduction The term ‘triple negative breast cancer’ (TNBC) is used to classify the 10% to 20% of all breast cancers that lack estrogen receptor (ER) and progesterone receptor (PR) expression as well as amplification of the human epidermal growth factor receptor 2 (HER2) [1]. Disease heterogeneity and the absence of well-defined molecular targets have made treatment of TNBC challenging. There is a major need to understand better the molecular basis of this type of breast cancer in order to develop effective therapeutic strategies [1]. In a previous study, we performed gene expression (GE) analyses and identified six distinct molecular TNBC subtypes with unique biological drivers [2], including one that was enriched for mesenchymalassociated genes termed mesenchymal-stem like (MSL). The MSL subtype is characterized by increased expression of genes related to transforming growth factor beta (TGF-β) signaling as well as pathways that play roles in extracellular matrix (ECM), focal adhesion, cell motility and cell differentiation [2]. Of note, TGF-β receptor type III (TβRIII) (gene symbol: TGFBR3) was among the differentially expressed TGF-β signaling components in the MSL subtype. The TGF-β signaling pathway has been implicated in cancer initiation and progression through tumor cell autonomous and non-autonomous signaling [3,4]. Initially identified as a tumor suppressor and then as a mediator of tumor progression, TGF-β signaling demonstrates diverse capabilities in cancer. The TGF-β pathway suppresses tumor growth through regulation of epithelial and stromal cell signaling [5]. Dysfunction of the pathway leads to carcinoma progression and metastasis [3]. While there has been significant focus on TGF-β receptor type I (TβRI) and TGF-β receptor type II (TβRII), research on TβRIII has lagged. Prior studies have demonstrated that TβRIII can regulate TGF-β signaling either via delivering TGF-β2 ligand to TβRII [6-9] or by binding to the cytoplasmic domain of TβRII, forming an active TβRI-TβRII signaling complex [10-13]. Currently, analysis of gene expression data sets generated from multiple cancer types indicates that TβRIII expression is decreased in highergrade cancers [14-17]. However, the role of TβRIII is controversial in breast cancer, since it has been reported that TβRIII can act as either a tumor suppressor or promoter in this cancer [18,19]. In the current study, we focused our investigations on the functional role of TβRIII in the MSL subtype of TNBC. We used a loss-of-function approach in representative MSL cell lines to demonstrate that TβRIII is required for maintenance of tumorigenicity in MSL TNBC cell lines and that regulation of integrin-α2 (gene symbol: ITGA2) expression is mechanistically involved in the observed phenotypes. This study demonstrates that TβRIII promotes the in vivo growth of a subset of TNBC and
Page 2 of 14
provides a pre-clinical rationale for consideration of TβRIII as a potential target for further discovery efforts.
Materials and Methods Cell culture
SUM159 cells (Asterand, Detroit, MI, USA) were maintained in (Dulbecco’s) Modified Eagle’s Medium: Nutrient Mixture F12 ((D)MEM-F12, GIBCO, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (GIBCO) and 0.5 μg/ml hydrocortisone. MDAMB-231 and MDA-MB-157 (ATTC, Manassas, VA, USA) were maintained in (D)MEM (GIBCO) supplemented with 10% FBS. Stable TβRIII-KD SUM159 cell lines were generated by lentiviral infection with virus carrying four independent short hairpin RNA (shRNA) clones (sequence-verified shRNA, pLKO.1-puro), (Sigma-Aldrich, St. Louis, MO, USA), Mission shRNA library #SHCLNG-NM_003243: clone#TRCN0000033433 (TβRIII-KD), clone#TRCN0000359000 (TβRIII-KD2), clone#TRCN0000359001 (TβRIII-KD3), and clone# TRCN0000359081 (TβRIII-KD4)) followed by puromycin selection (Invitrogen-Life Technology, Inc, Carlsbad, CA, USA). MDA-MB-231 and MDA-MB-157 were stably infected with clone# TRCN0000033433. Integrin-α2 was stably knocked down in TβRIII-KD MSL cells using lentiviral particles carrying shRNA to integrin-α2 (α2KD) (Sigma-Aldrich, Mission shRNA validated library, #SHCLNG-NM_002203, clone#TRCN0000308081). Three-dimensional culture assay
The wells in 48-well plates were coated with 50 μl of growth factor reduced BD Matrigel (BD Biosciences #356231, San Jose, CA, USA) and allowed to polymerize at 37°C for 15 minutes. Then, 5 x 105 cells were resuspended in 200 μl of growth factor reduced BD Matrigel and plated onto the matigel-coated wells. Plates were incubated for 30 minutes after which 1 ml of media was added to the top of the matrigel. Media was replenished every 48 hours. Images were taken at day six. Quantification of the images was performed using Fiji Software. Cell proliferation assays Cell counts
Cells were plated into six-well plates at a density of 1.25 x 105 cells/well. The following day cells were treated with 1 ng/ml TGF-β1 (R&D Systems, #102-B1, Minneapolis, MN, USA) and TGF-β2 (R&D Systems, #102-B2). After 72 hours treatment with TGF-β, viable cells were counted. 3
H-Thymidine incorporation assay
A total of 2.5 × 104 cells were plated in a 24-well dish and allowed to grow overnight. The next day the medium was aspirated and replaced with complete medium containing +/−TGF-β1 or TGF-β2 (1 ng/ml). The cells were
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
then subjected to [3H] thymidine incorporation assay as previously described [20]. Migration and invasion assays Magnetic attachable stencils migration assays
This migration method serves as a more reproducible alternative to the scratch assay. The use of magnetic force to attach stencil to the multi-well plates is a new strategy that creates defined and reproducible cell-free voids for quantitation of cell migration and has been well characterized and described by Ashby et al. [21]. Magnetic attachable stencils (MAtS) were attached to the surfaces of each well of a 12well plate by placing a platform with magnets underneath and in direct contact with the 12-well plate. Cells were then plated in triplicate at 7.5 x 105 cells per well around the MAtS in serum-free media. The next day the MAtS were removed and cells were treated with 1 ng/ml TGF-β1 (R&D Systems, #102-B1) and 1 ng/ml TGF-β2 (R&D Systems, #102-B2). Gap closure was quantified (Tscratch software) at both 0 and 24 hours and percent of closure determined with the following equation: percent of closure = average of ((gap area: 0 hour) – (gap area: 24 hours))/(gap area: 0 hour) using images from 12 different microscopic fields per well (4X magnification).
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Mice were monitored weekly for tumor growth. Tumor measurements were performed once a week for five weeks after palpable tumors formed. Tumor volume was measured at the indicated times with calipers, and tumor volumes were calculated as width2 x length/2. All mouse experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC). Luciferase reporter assay
Cells were seeded at a density of 2 X 104 cells/well in 12well tissue culture plates. The following day, the cells were transiently transfected using Transfectin lipid reagent following the manufacturer’s protocol (Bio-Rad #170-3351, Hercules, CA, USA). Cells were transfected with 1.5 μg 3TP-Lux [22] or CAGA(9)-Luc [23]. pRL-CMV-renilla (Promega #E2261, Madison, WI, USA) was co-transfected and used as an internal control to correct for transfection efficiency. Eighteen hours after transfection, cells were treated with 1 ng/ml TGF-β1 or TGFβ-2 (R&D Systems, #102-B1 and #102-B2, respectively). Twenty-four hours after TGF-β treatment, cells were harvested and assayed for promoter specific luciferase activity using a Dual-Luciferase Reporter Assay System (Promega #E1910) according to the manufacturer’s protocol. Luciferase activity was measured using a BD/Pharmigen Monolight 3010 luminometer.
Transwell assays
Migrations (Costar, #3422, Tewksbury, MA, USA) were conducted by plating 2.5 x 104 cells in the top of the transwell and media with 10% FBS in the bottom of the well to act as a chemoattractant. Cells were fixed in 4% paraformaldehyde and stained with 4′, 6-diamidino-2-phenylindole (DAPI). Quantification was performed by taking pictures of multiple regions of the membrane after which cells’ nuclei were counted using Metamorph software. The same migration assay was used to measure blocked integrin-α2 function. The TβRIII-KD cells were incubated for 30 minutes with integrin-α2 blocking antibody (Abcam, #ab24697, Cambridge, MA, USA) washed two times with PBS and plated in the top of the transwell. Invasion assays were conducted by plating 5 x 105 cells in serum-free media in the upper chamber, pre-coated with growth factor reduced matrigel. In the bottom chamber (D)MEM with 10% FBS was used as a chemoattractant (BD Biosciences, #354483). Cells that had invaded through the matrigel were fixed in 4% paraformaldehyde and stained using DAPI. Quantification of cells that invaded into the matrigel was performed using the same protocol as described for the transwell assays. Xenograft tumor studies
One milllion cells embeded in collagen were implanted into the number four gland of six- to eight-week-old female athymic nude- Foxn1nu/nu mice (purchased from Harlan Sprague- Dawley, Inc., Indianapolis, IN, USA).
RNA preparation and quantitative PCR
RNA was isolated and purified using an RNeasy Mini Kit and an RNase-Free DNase Set (Qiagen, Valencia, CA, USA). A total of 750 μg of RNA was used to synthesize cDNA using Superscript III reverse transcriptase as described by the manufacturer (Invitrogen). Bio-Rad iCycler and CFX96 machines were used for qPCR employing Power SYBR Green (Applied Biosystems, Carlsbad, CA, USA) or SsoAdvanced SYBR Green Supermix (Bio-Rad), respectively. Ct values were normalized to GAPDH for statistical analyses. Primer sequences are available in Additional file 1. Immunoblotting
Standard protein preparation and electrophoresis procedures were used as described [4]. Western membranes were blocked in 5% milk and incubated with primary antibody overnight. The antibody list with concentrations and the catalog numbers are available in Additional file 1. Flow cytometry
Cells were detached using Accutase (Life Technologies), pelleted, washed and counted. One million cells were incubated with TβRIII antibody (Cell Signaling, #5544, Danvers, MA, USA) for 30 minutes, washed, and then incubated at 4°C with Alexa Fluor 488 conjugated secondary antibody (Life Techologies, #A11034) for 30 minutes. One million cells were labeled with fluorescence-conjugated integrin-α2
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
antibody (BioLegend, #314308, San Diego, CA, USA) for 30 minutes at 4°C. Cells were washed three times then analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest Pro software. Data were analyzed with FlowJo software (Tree Star). Microarray gene expression analysis Public database analysis
Human tissue and cell line microarray datasets were analyzed using GeneSpring GX 12.0 microarray analysis software (Agilent). Previously published TNBC gene expression profiles (n = 587 patients) [2] consisting of publicly available microarray data sets (the GEO registration numbers are referenced in Additional file 1) were obtained and processed as previously described and were in compliance with ethical requirements [2]. Comparisons between expression of TGFBR3 and ITGA2 for different TNBC subtypes were performed in R 3.0.1 [24] using the t test function for paired two-tailed Student’s t-tests and graphically represented using ggplot2 [25]. In vitro three-dimensional culture analysis
vRNA was extracted from SUM159 three-dimensional culture samples and hybridized to the human gene 1.0ST array, scanned with Affymetrix using AGCC v. 3.2.4 and then analyzed in R 3.0.1 using the oligo package. Samples were normalized with the RMA algorithm, genes were annotated with the pd.hugene.1.0.st.v1 package, and differential gene expression analysis was conducted using the limma package. The three-dimensional culture microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus [26] and are accessible through GEO Series accession number GSE54756 [27]. Statistical analysis
All data were analyzed using the unpaired two-tailed Student’s t test (GraphPad Prism 5). Error bars show mean ± SEM. A two-sided P value less than 0.05 was considered significantly different.
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of the TNBC subtypes (Additional file 2: Figure S1). Similarly, analysis of TGFBR3 gene expression across a panel of TNBC cell lines, representative of the various subtypes, demonstrates that the M and MSL subtypes have relatively higher levels of TGFBR3 mRNA (Figure 1C-D). These findings were validated by qPCR (Figure 1E) and immunoblot analyses for TβRIII protein levels (Figure 1F). Although the TNBC M subtype cell lines also showed increased levels of TβRIII expression, we focused our studies of this receptor on the MSL subtype as their expression is more consistent with human datasets (Figure 1A-B). Knockdown of TβRIII in MSL TNBC cells leads to decreased tumorigenicity in vivo
In order to determine the significance of the TβRIII expression in MSL TNBC cell behavior, we knocked down TβRIII in MSL cells and performed orthotopic xenograft tumor studies. We used a panel of four shRNA expression vectors to optimize TβRIII knockdown, as validated by immunoblot and flow cytometry analyses (Figure 2A-C). We utilized immunocompromised nude mice to establish orthotopic xenograft tumors from cell lines representing the MSL subtype of TNBC with and without TβRIII knockdown. Initially we tested SUM159 cells with two shRNA expression vectors (TβRIII-KD and TβRIII-KD4) to eliminate off target effects of the shRNA (Additional file 2: Figure S2). After establishing that both expression vectors resulted in a similar phenotype, we used a single shRNA (TβRIII-KD) in all subsequent experiments across three MSL cell lines. Knockdown of TβRIII in the SUM159 and MDA-MB-231 MSL cell lines significantly decreased xenograft tumor growth (Figure 2DE). MDA-MB-157 showed inconsistent results (Additional file 2: Figure S3A) and after further investigation we discovered that the TβRIII-KD tumors expressed TβRIII (Additional file 2: Figure S3B). Thus, either there was a selection against the knockdown in vivo and, therefore, the tumor cells expressed TβRIII, or there was a small subpopulation of MDA-MB-157 cells at the start of the experiment that retained expression and seeded the tumor growth. Regardless, both explanations provide further evidence for the tumor-promoting effect of TβRIII.
Results Human mesenchymal stem-like triple negative breast tumors and representative cell lines have increased TβRIII expression
Using a gene expression data set generated from 587 TNBC tumors, we examined the relative mRNA levels of TGF-β receptors and ligands across subtypes of TNBC. We observed elevated expression of TGFBR3 in basal-like1 (BL1), mesenchymal (M) and MSL tumors (Figure 1A). The highest relative level of TGFBR3 expression was in the MSL subtype (Figure 1B). Average probe intensities for the TGFβ receptors I and II as well as TGF β ligands 1 and 3 were also elevated in the MSL subtype in comparison to the rest
Knockdown of TβRIII in MSL cell lines does not affect cell proliferation or viability
Since TβRIII-KD markedly decreased the tumorigenic potential of mesenchymal TNBC cells, we further explored whether this was due to a proliferation defect. TβRIII can bind to all TGF-β ligands but with highest affinity for TGF-β2 [29,30]; therefore, cells were treated with TGF-β2 in addition to TGF-β1. Both controls and TβRIII-KD MSL cell lines responded similarly to the ligands (Figure 3A-B). TβRIII-KD did not alter the proliferation rates of MSL cell lines (SUM159, MDA-MB-231 and MDA-MB-157) by live cell counts (Figure 3A) or
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
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(B) TGFBR3 Average probe intensities
(A) TGFB1 TGFB2 TGFB3 TGFBR1 TGFBR2 TGFBR3 Intensities (Log 2)
BL1 /BL2
M
CAL148
MFM223
MDA453
MDA157
SUM159
CAL51
MDA231
CAL120
HCC38
HCC70
CAL851
TGFBR3
Intensities (Log 2)
0.0 -0.5
BL1
-1.0
M
IM
BL2
MSL
LAR
4
** 3
**
ns
2
1
0
-1
BL1/BL2
M
MSL
LAR
(F)
20000 18000
BL1/BL2
M
MSL
LAR
12000 10000 8000 6000
MDA453 CAL148 MFM223 SUM185
14000
CAL51 BT549 CAL120 MDA157 MDA231 SUM159
HCC38 CAL851 HCC70 HDQP1
16000
T RIII (300kDa)
4000 2000 0
HCC38 CAL851 HCC70 HDQP1 CAL51 BT549 CAL120 MDA157 MDA231 SUM159 MDA453 CAL148 MFM223 SUM185
TGFBR3 Mean fold change mRNA expression (2-ΔΔCt)
(E)
***
0.5
(D)
LAR
MSL
*** *** ***
1.0
TGFBR3 Average probe intensities
(C)
** 1.5
BL1/BL2
M
MSL
T RIII (110kDa) Actin
LAR
Figure 1 TGFBR3 gene expression levels are elevated in the mesenchymal stem-like (MSL) subtype of TNBC. A) Heat map representation of gene expression for 587 TNBC tumors for each TGF-β ligand and receptor. B) Quantification of average TGFBR3 gene expression across TNBC tumor subtypes, average based on individual TNBC tumor probe intensity values (**P = 0.004; ***P
RESEARCH ARTICLE
Open Access
Transforming growth factor beta receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer Bojana Jovanović1, J Scott Beeler2, Michael W Pickup1, Anna Chytil1, Agnieszka E Gorska1, William J Ashby3, Brian D Lehmann2, Andries Zijlstra4, Jennifer A Pietenpol1,2 and Harold L Moses1,4*
Abstract Introduction: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-β) pathway-associated genes relative to other subtypes, including the TGF-β receptor type III (TβRIII). We hypothesize that TβRIII is tumor promoter in mesenchymal-stem like TNBC cells. Methods: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TβRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TβRIII (TβRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TβRIII expression patterns across all TNBC subtypes. Results: TβRIII was the most differentially expressed TGF-β signaling gene in the MSL subtype. Silencing TβRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TβRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TβRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TβRIII-KD. Stable knockdown of integrin-α2 in TβRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. Conclusions: We have found that TβRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TβRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TβRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TβRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.
* Correspondence: [email protected] 1 Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, 2220 Pierce Avenue, 698 Preston Research Building, Nashville, TN 37232, USA 4 Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, 1161 21st Avenue South, C-3321A Medical Center North, Nashville, TN 37232, USA Full list of author information is available at the end of the article © 2014 Jovanović et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
Introduction The term ‘triple negative breast cancer’ (TNBC) is used to classify the 10% to 20% of all breast cancers that lack estrogen receptor (ER) and progesterone receptor (PR) expression as well as amplification of the human epidermal growth factor receptor 2 (HER2) [1]. Disease heterogeneity and the absence of well-defined molecular targets have made treatment of TNBC challenging. There is a major need to understand better the molecular basis of this type of breast cancer in order to develop effective therapeutic strategies [1]. In a previous study, we performed gene expression (GE) analyses and identified six distinct molecular TNBC subtypes with unique biological drivers [2], including one that was enriched for mesenchymalassociated genes termed mesenchymal-stem like (MSL). The MSL subtype is characterized by increased expression of genes related to transforming growth factor beta (TGF-β) signaling as well as pathways that play roles in extracellular matrix (ECM), focal adhesion, cell motility and cell differentiation [2]. Of note, TGF-β receptor type III (TβRIII) (gene symbol: TGFBR3) was among the differentially expressed TGF-β signaling components in the MSL subtype. The TGF-β signaling pathway has been implicated in cancer initiation and progression through tumor cell autonomous and non-autonomous signaling [3,4]. Initially identified as a tumor suppressor and then as a mediator of tumor progression, TGF-β signaling demonstrates diverse capabilities in cancer. The TGF-β pathway suppresses tumor growth through regulation of epithelial and stromal cell signaling [5]. Dysfunction of the pathway leads to carcinoma progression and metastasis [3]. While there has been significant focus on TGF-β receptor type I (TβRI) and TGF-β receptor type II (TβRII), research on TβRIII has lagged. Prior studies have demonstrated that TβRIII can regulate TGF-β signaling either via delivering TGF-β2 ligand to TβRII [6-9] or by binding to the cytoplasmic domain of TβRII, forming an active TβRI-TβRII signaling complex [10-13]. Currently, analysis of gene expression data sets generated from multiple cancer types indicates that TβRIII expression is decreased in highergrade cancers [14-17]. However, the role of TβRIII is controversial in breast cancer, since it has been reported that TβRIII can act as either a tumor suppressor or promoter in this cancer [18,19]. In the current study, we focused our investigations on the functional role of TβRIII in the MSL subtype of TNBC. We used a loss-of-function approach in representative MSL cell lines to demonstrate that TβRIII is required for maintenance of tumorigenicity in MSL TNBC cell lines and that regulation of integrin-α2 (gene symbol: ITGA2) expression is mechanistically involved in the observed phenotypes. This study demonstrates that TβRIII promotes the in vivo growth of a subset of TNBC and
Page 2 of 14
provides a pre-clinical rationale for consideration of TβRIII as a potential target for further discovery efforts.
Materials and Methods Cell culture
SUM159 cells (Asterand, Detroit, MI, USA) were maintained in (Dulbecco’s) Modified Eagle’s Medium: Nutrient Mixture F12 ((D)MEM-F12, GIBCO, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (GIBCO) and 0.5 μg/ml hydrocortisone. MDAMB-231 and MDA-MB-157 (ATTC, Manassas, VA, USA) were maintained in (D)MEM (GIBCO) supplemented with 10% FBS. Stable TβRIII-KD SUM159 cell lines were generated by lentiviral infection with virus carrying four independent short hairpin RNA (shRNA) clones (sequence-verified shRNA, pLKO.1-puro), (Sigma-Aldrich, St. Louis, MO, USA), Mission shRNA library #SHCLNG-NM_003243: clone#TRCN0000033433 (TβRIII-KD), clone#TRCN0000359000 (TβRIII-KD2), clone#TRCN0000359001 (TβRIII-KD3), and clone# TRCN0000359081 (TβRIII-KD4)) followed by puromycin selection (Invitrogen-Life Technology, Inc, Carlsbad, CA, USA). MDA-MB-231 and MDA-MB-157 were stably infected with clone# TRCN0000033433. Integrin-α2 was stably knocked down in TβRIII-KD MSL cells using lentiviral particles carrying shRNA to integrin-α2 (α2KD) (Sigma-Aldrich, Mission shRNA validated library, #SHCLNG-NM_002203, clone#TRCN0000308081). Three-dimensional culture assay
The wells in 48-well plates were coated with 50 μl of growth factor reduced BD Matrigel (BD Biosciences #356231, San Jose, CA, USA) and allowed to polymerize at 37°C for 15 minutes. Then, 5 x 105 cells were resuspended in 200 μl of growth factor reduced BD Matrigel and plated onto the matigel-coated wells. Plates were incubated for 30 minutes after which 1 ml of media was added to the top of the matrigel. Media was replenished every 48 hours. Images were taken at day six. Quantification of the images was performed using Fiji Software. Cell proliferation assays Cell counts
Cells were plated into six-well plates at a density of 1.25 x 105 cells/well. The following day cells were treated with 1 ng/ml TGF-β1 (R&D Systems, #102-B1, Minneapolis, MN, USA) and TGF-β2 (R&D Systems, #102-B2). After 72 hours treatment with TGF-β, viable cells were counted. 3
H-Thymidine incorporation assay
A total of 2.5 × 104 cells were plated in a 24-well dish and allowed to grow overnight. The next day the medium was aspirated and replaced with complete medium containing +/−TGF-β1 or TGF-β2 (1 ng/ml). The cells were
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
then subjected to [3H] thymidine incorporation assay as previously described [20]. Migration and invasion assays Magnetic attachable stencils migration assays
This migration method serves as a more reproducible alternative to the scratch assay. The use of magnetic force to attach stencil to the multi-well plates is a new strategy that creates defined and reproducible cell-free voids for quantitation of cell migration and has been well characterized and described by Ashby et al. [21]. Magnetic attachable stencils (MAtS) were attached to the surfaces of each well of a 12well plate by placing a platform with magnets underneath and in direct contact with the 12-well plate. Cells were then plated in triplicate at 7.5 x 105 cells per well around the MAtS in serum-free media. The next day the MAtS were removed and cells were treated with 1 ng/ml TGF-β1 (R&D Systems, #102-B1) and 1 ng/ml TGF-β2 (R&D Systems, #102-B2). Gap closure was quantified (Tscratch software) at both 0 and 24 hours and percent of closure determined with the following equation: percent of closure = average of ((gap area: 0 hour) – (gap area: 24 hours))/(gap area: 0 hour) using images from 12 different microscopic fields per well (4X magnification).
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Mice were monitored weekly for tumor growth. Tumor measurements were performed once a week for five weeks after palpable tumors formed. Tumor volume was measured at the indicated times with calipers, and tumor volumes were calculated as width2 x length/2. All mouse experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC). Luciferase reporter assay
Cells were seeded at a density of 2 X 104 cells/well in 12well tissue culture plates. The following day, the cells were transiently transfected using Transfectin lipid reagent following the manufacturer’s protocol (Bio-Rad #170-3351, Hercules, CA, USA). Cells were transfected with 1.5 μg 3TP-Lux [22] or CAGA(9)-Luc [23]. pRL-CMV-renilla (Promega #E2261, Madison, WI, USA) was co-transfected and used as an internal control to correct for transfection efficiency. Eighteen hours after transfection, cells were treated with 1 ng/ml TGF-β1 or TGFβ-2 (R&D Systems, #102-B1 and #102-B2, respectively). Twenty-four hours after TGF-β treatment, cells were harvested and assayed for promoter specific luciferase activity using a Dual-Luciferase Reporter Assay System (Promega #E1910) according to the manufacturer’s protocol. Luciferase activity was measured using a BD/Pharmigen Monolight 3010 luminometer.
Transwell assays
Migrations (Costar, #3422, Tewksbury, MA, USA) were conducted by plating 2.5 x 104 cells in the top of the transwell and media with 10% FBS in the bottom of the well to act as a chemoattractant. Cells were fixed in 4% paraformaldehyde and stained with 4′, 6-diamidino-2-phenylindole (DAPI). Quantification was performed by taking pictures of multiple regions of the membrane after which cells’ nuclei were counted using Metamorph software. The same migration assay was used to measure blocked integrin-α2 function. The TβRIII-KD cells were incubated for 30 minutes with integrin-α2 blocking antibody (Abcam, #ab24697, Cambridge, MA, USA) washed two times with PBS and plated in the top of the transwell. Invasion assays were conducted by plating 5 x 105 cells in serum-free media in the upper chamber, pre-coated with growth factor reduced matrigel. In the bottom chamber (D)MEM with 10% FBS was used as a chemoattractant (BD Biosciences, #354483). Cells that had invaded through the matrigel were fixed in 4% paraformaldehyde and stained using DAPI. Quantification of cells that invaded into the matrigel was performed using the same protocol as described for the transwell assays. Xenograft tumor studies
One milllion cells embeded in collagen were implanted into the number four gland of six- to eight-week-old female athymic nude- Foxn1nu/nu mice (purchased from Harlan Sprague- Dawley, Inc., Indianapolis, IN, USA).
RNA preparation and quantitative PCR
RNA was isolated and purified using an RNeasy Mini Kit and an RNase-Free DNase Set (Qiagen, Valencia, CA, USA). A total of 750 μg of RNA was used to synthesize cDNA using Superscript III reverse transcriptase as described by the manufacturer (Invitrogen). Bio-Rad iCycler and CFX96 machines were used for qPCR employing Power SYBR Green (Applied Biosystems, Carlsbad, CA, USA) or SsoAdvanced SYBR Green Supermix (Bio-Rad), respectively. Ct values were normalized to GAPDH for statistical analyses. Primer sequences are available in Additional file 1. Immunoblotting
Standard protein preparation and electrophoresis procedures were used as described [4]. Western membranes were blocked in 5% milk and incubated with primary antibody overnight. The antibody list with concentrations and the catalog numbers are available in Additional file 1. Flow cytometry
Cells were detached using Accutase (Life Technologies), pelleted, washed and counted. One million cells were incubated with TβRIII antibody (Cell Signaling, #5544, Danvers, MA, USA) for 30 minutes, washed, and then incubated at 4°C with Alexa Fluor 488 conjugated secondary antibody (Life Techologies, #A11034) for 30 minutes. One million cells were labeled with fluorescence-conjugated integrin-α2
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
antibody (BioLegend, #314308, San Diego, CA, USA) for 30 minutes at 4°C. Cells were washed three times then analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest Pro software. Data were analyzed with FlowJo software (Tree Star). Microarray gene expression analysis Public database analysis
Human tissue and cell line microarray datasets were analyzed using GeneSpring GX 12.0 microarray analysis software (Agilent). Previously published TNBC gene expression profiles (n = 587 patients) [2] consisting of publicly available microarray data sets (the GEO registration numbers are referenced in Additional file 1) were obtained and processed as previously described and were in compliance with ethical requirements [2]. Comparisons between expression of TGFBR3 and ITGA2 for different TNBC subtypes were performed in R 3.0.1 [24] using the t test function for paired two-tailed Student’s t-tests and graphically represented using ggplot2 [25]. In vitro three-dimensional culture analysis
vRNA was extracted from SUM159 three-dimensional culture samples and hybridized to the human gene 1.0ST array, scanned with Affymetrix using AGCC v. 3.2.4 and then analyzed in R 3.0.1 using the oligo package. Samples were normalized with the RMA algorithm, genes were annotated with the pd.hugene.1.0.st.v1 package, and differential gene expression analysis was conducted using the limma package. The three-dimensional culture microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus [26] and are accessible through GEO Series accession number GSE54756 [27]. Statistical analysis
All data were analyzed using the unpaired two-tailed Student’s t test (GraphPad Prism 5). Error bars show mean ± SEM. A two-sided P value less than 0.05 was considered significantly different.
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of the TNBC subtypes (Additional file 2: Figure S1). Similarly, analysis of TGFBR3 gene expression across a panel of TNBC cell lines, representative of the various subtypes, demonstrates that the M and MSL subtypes have relatively higher levels of TGFBR3 mRNA (Figure 1C-D). These findings were validated by qPCR (Figure 1E) and immunoblot analyses for TβRIII protein levels (Figure 1F). Although the TNBC M subtype cell lines also showed increased levels of TβRIII expression, we focused our studies of this receptor on the MSL subtype as their expression is more consistent with human datasets (Figure 1A-B). Knockdown of TβRIII in MSL TNBC cells leads to decreased tumorigenicity in vivo
In order to determine the significance of the TβRIII expression in MSL TNBC cell behavior, we knocked down TβRIII in MSL cells and performed orthotopic xenograft tumor studies. We used a panel of four shRNA expression vectors to optimize TβRIII knockdown, as validated by immunoblot and flow cytometry analyses (Figure 2A-C). We utilized immunocompromised nude mice to establish orthotopic xenograft tumors from cell lines representing the MSL subtype of TNBC with and without TβRIII knockdown. Initially we tested SUM159 cells with two shRNA expression vectors (TβRIII-KD and TβRIII-KD4) to eliminate off target effects of the shRNA (Additional file 2: Figure S2). After establishing that both expression vectors resulted in a similar phenotype, we used a single shRNA (TβRIII-KD) in all subsequent experiments across three MSL cell lines. Knockdown of TβRIII in the SUM159 and MDA-MB-231 MSL cell lines significantly decreased xenograft tumor growth (Figure 2DE). MDA-MB-157 showed inconsistent results (Additional file 2: Figure S3A) and after further investigation we discovered that the TβRIII-KD tumors expressed TβRIII (Additional file 2: Figure S3B). Thus, either there was a selection against the knockdown in vivo and, therefore, the tumor cells expressed TβRIII, or there was a small subpopulation of MDA-MB-157 cells at the start of the experiment that retained expression and seeded the tumor growth. Regardless, both explanations provide further evidence for the tumor-promoting effect of TβRIII.
Results Human mesenchymal stem-like triple negative breast tumors and representative cell lines have increased TβRIII expression
Using a gene expression data set generated from 587 TNBC tumors, we examined the relative mRNA levels of TGF-β receptors and ligands across subtypes of TNBC. We observed elevated expression of TGFBR3 in basal-like1 (BL1), mesenchymal (M) and MSL tumors (Figure 1A). The highest relative level of TGFBR3 expression was in the MSL subtype (Figure 1B). Average probe intensities for the TGFβ receptors I and II as well as TGF β ligands 1 and 3 were also elevated in the MSL subtype in comparison to the rest
Knockdown of TβRIII in MSL cell lines does not affect cell proliferation or viability
Since TβRIII-KD markedly decreased the tumorigenic potential of mesenchymal TNBC cells, we further explored whether this was due to a proliferation defect. TβRIII can bind to all TGF-β ligands but with highest affinity for TGF-β2 [29,30]; therefore, cells were treated with TGF-β2 in addition to TGF-β1. Both controls and TβRIII-KD MSL cell lines responded similarly to the ligands (Figure 3A-B). TβRIII-KD did not alter the proliferation rates of MSL cell lines (SUM159, MDA-MB-231 and MDA-MB-157) by live cell counts (Figure 3A) or
Jovanović et al. Breast Cancer Research 2014, 16:R69 http://breast-cancer-research.com/content/16/4/R69
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(B) TGFBR3 Average probe intensities
(A) TGFB1 TGFB2 TGFB3 TGFBR1 TGFBR2 TGFBR3 Intensities (Log 2)
BL1 /BL2
M
CAL148
MFM223
MDA453
MDA157
SUM159
CAL51
MDA231
CAL120
HCC38
HCC70
CAL851
TGFBR3
Intensities (Log 2)
0.0 -0.5
BL1
-1.0
M
IM
BL2
MSL
LAR
4
** 3
**
ns
2
1
0
-1
BL1/BL2
M
MSL
LAR
(F)
20000 18000
BL1/BL2
M
MSL
LAR
12000 10000 8000 6000
MDA453 CAL148 MFM223 SUM185
14000
CAL51 BT549 CAL120 MDA157 MDA231 SUM159
HCC38 CAL851 HCC70 HDQP1
16000
T RIII (300kDa)
4000 2000 0
HCC38 CAL851 HCC70 HDQP1 CAL51 BT549 CAL120 MDA157 MDA231 SUM159 MDA453 CAL148 MFM223 SUM185
TGFBR3 Mean fold change mRNA expression (2-ΔΔCt)
(E)
***
0.5
(D)
LAR
MSL
*** *** ***
1.0
TGFBR3 Average probe intensities
(C)
** 1.5
BL1/BL2
M
MSL
T RIII (110kDa) Actin
LAR
Figure 1 TGFBR3 gene expression levels are elevated in the mesenchymal stem-like (MSL) subtype of TNBC. A) Heat map representation of gene expression for 587 TNBC tumors for each TGF-β ligand and receptor. B) Quantification of average TGFBR3 gene expression across TNBC tumor subtypes, average based on individual TNBC tumor probe intensity values (**P = 0.004; ***P
